草甘膦单克隆抗体的制备及酶联免疫分析方法的建立

Preparation of glyphosate monoclonal antibody and establishment of an enzyme linked immunosorbent assay

中文关键词:  草甘膦  完全抗原  单克隆抗体  间接竞争酶联免疫分析法

英文关键词:glyphosate  complete antigen  monoclonal antibody  enzyme linked immunosorbent assay

中文摘要:

      目的 制备出草甘膦(glyphosate, GLY)单克隆抗体, 建立间接竞争酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)快速检测茶叶中GLY的方法。方法 首先合成GLY完全抗原(包被原和免疫原), 通过免疫小鼠成功制备出GLY单克隆抗体。根据ELISA的检测流程, 采用棋盘法确定最佳包被抗原和抗体的稀释倍数, 确定最佳包被的温度和时间, 并确定最佳加入一抗、二抗后反应时间, 建立检测方法并对其性能进行评价。结果 GLY包被抗原和抗体的稀释倍数为1:2000, 包被温度为37℃、包被时间为90 min, 加入一抗、二抗后反应时间为60 min。该方法的线性方程为Y=-0.2353X+0.6539 (r2=0.9871), 半抑制浓度(50% inhibiting concentration, IC50)为4.508 ng/mL, 检出限为1.18 ng/mL。变异系数均在10%以下, 与异菌脲、多菌灵、三唑磷、甲基对硫磷、噻菌灵这5种标准品的交叉反应率均低于0.03%, 在茶叶中加标回收率为90.86%~110.35%, 且相对标准偏差均小于10%。结论 该方法具有较高的灵敏度和特异性, 可用于茶叶样本中GLY残留的快速检测。

英文摘要:

      Objective To prepare monoclonal antibody to glyphosate (GLY), and establish a method for rapid detection of GLY in tea leaves by indirect competition enzyme linked immunosorbent assay (ELISA). Methods Firstly, GLY complete antigen (coated and immunogen) was synthesized, and GLY monoclonal antibody was successfully prepared by immunizing mice. According to the detection process of ELISA, the dilution ratio of the optimal coated antigen and antibody was determined, the temperature and time of the optimal coating were determined, and the reaction time after adding the optimal primary and secondary antibodies was determined, so as to establish the detection method and evaluate its performance. Results The dilution of GLY coated antigen and antibody was 1:2000, the coating temperature was 37℃, the coating time was 90 min, and the reaction time was 60 min after adding the primary antibody and the secondary antibody. The linear equation of this method was Y=-0.2353X+0.6539 (r2=0.9871), the 50% inhibiting concentration (IC50) was 4.508 ng/mL, and the limit of detection was 1.18 ng/mL. The coefficient of variation was below 10%, and the cross reaction rate with the 5 kinds of standards of urerea, polyazin, triazole, methyl and thiazine was less than 0.03%, the recovery rate of labeling in tea was between 90.86% and 110.35%, and the relative standard deviations were less than 10%. Conclusion This method has high sensitivity and specificity and can be used for rapid detection of GLY residues in tea samples.